The present invention generally relates to a thermal cycler for titration plates and more particularly pertains to a device that is capable of controlling the temperature of the contents of individually selected sample wells within a multi-well titration plate.
The Polymerase Chain Reaction (PCR) process effects the replication of long-chain DNA molecules and is today an essential tool in genetics and molecular biology. It is the central component in diagnostics, therapeutics and genomics involving DNA amplification. The process is commenced with a denaturing step typically at 95C at which point strands of the DNA double helix in a solution are separated. After time to equilibrate, the temperature is rapidly reduced to the annealing temperature (typically, 50 to 65C) where primers hybridize to the two separated DNA strands. Thus attached, the primers allow the formation of new DNA at the optimum synthesis temperature (typically, 72C), where the chain-length of the DNA produced depends on the time held at this temperature. To double the molecules produced, the temperature cycle is repeated. Thus, a single molecule of DNA will result in 34 billion copies after 35 cycles (235)
Titration plates are commonly employed in laboratory work of various disciplines to store multiple samples, typically in a closely spaced 8xc3x9712 pattern of sample wells. The titration plate is often of monolithic construction and may comprise a single injection molding of a chemically inert plastic material. Each individual well extends downwardly from the flat top face of the plate, is typically cylindrical in cross-section and is provided with a flat, U-shaped or V-shaped bottom to support a sample volume of 1 ml.
Titration plates offer a convenient means for processing large numbers of samples and are used to subject samples to PCR and DNA amplification. A distinct disadvantage inherent in the use of a titration plate as described above for such application is that heretofore thermal cyclers have typically utilized a single temperature block such that all samples contained in a single titration plate must execute the same PCR program simultaneously. An additional disadvantage is inherent in the fact that single temperature block type devices may be subject to a temperature gradient within the block which may adversely affect the process.
A simple hot plate fulfills the most fundamental requirements while the more sophisticated heating devices have included features that endeavor to maintain as uniform a temperature as possible throughout the entire array of samples contained in a titration plate. Additionally, heating devices are known that subject the entire array of sample wells in a titration plate to a repetition of prescribed temperature gradients as is useful for PCR.
The prior art is devoid of a device that is capable of subjecting selected individual sample wells in a titration plate to PCR and DNA amplification techniques, independent of the temperatures of neighboring or unselected wells.
The present invention provides a heating apparatus that is capable of controlling the temperature of individual sample wells in a titration plate without affecting the temperature of neighboring sample wells. Moreover, the device of the present invention is capable of simultaneously subjecting individual sample wells of a titration plate to different temperatures and different rates of temperature change.
A programmable controller is employed to control the operation of each heating and cooling mechanism associated with each sample well. The use of a temperature sensor associated with each sample well that feeds temperature data back to the controller allows for more precise control of the temperature to yield high PCR efficiency. Different PCR temperature programs (cycles) or experiments can thereby be exercised in different wells of the same plate at the same time.
Preferred embodiments of the present invention may include an array of sleeves that are arranged and dimensioned to individually receive each of the sample wells of a titration plate placed thereover. Such sleeves may serve to direct or conduct heat to the well received therein and may optionally be relied upon to conduct heat away from the vial when not in the heating mode. Alternatively, the sleeves may be relied upon to merely properly position sample wells inserted thereinto relative to a source of conducted, convected or radiated heat. As a further alternative, the selective heating may be accomplished without the use of individual well receiving sleeves.
In a preferred embodiment, an array of thermally conductive sleeves extend upwardly from a cold plate which serves to conduct heat away from each sample well via the corresponding sleeve. Each sleeve is additionally fitted with an individually controllable heating element. By energizing such heating element, the thermally conductive sleeve conducts heat to the corresponding sample well to heat the material contained therein. Adjacent sample wells are unaffected by the heat generated by the energized heating element and continue to be maintained in their original state by virtue of their continued interconnection to the cold plate via their corresponding sleeves. Optionally, the sleeve is physically disconnected from the cold plate upon energization of the corresponding heating element to minimize heat loss and thereby expedite the heating process. A programmable controller is employed to enable an operator to select those heating elements which are to be energized.
In alternative embodiments, the exterior surface of each sample well is coated with a resistive material and the sleeve serves to conduct electricity thereto. As a result, heating is effected on the well itself. Alternatively, each sleeve is in direct contact with an individually controllable Peltier-effect device with which both the heating as well as cooling of each well is accomplished. As a further alternative, a source of radiant energy such as a laser is focused on each well wherein selective energization thereof serves to heat selected sample wells. Finally, the sleeve may be relied upon to direct a flow of heated fluid at each well to effect a heating thereof.
In a further alternative embodiment of the present invention, variable thermal contact with a cold plate is effected by bimetallic elements. In its deactivated state, the bimetallic element conducts heat from the sample to the cold plate. As the heating element is energized, the heat is transferred to both the sample as well as the bimetallic element which causes the later to deflect thereby breaking thermal contact with the cold plate. A shape memory material such as Nitinol can be substituted for the bimetallic element.
In any of the various embodiments of the present invention, separate temperature sensors may be associated with each individual sample well to provide feedback to the controller. Alternatively, a sensor mass may be associated with each sleeve to effect temperature measurement feedback for the thermal control. By appropriate adjustment of the sensor mass and its thermal resistance to the sleeve, its temperature can be shown to be dynamically equivalent to the solution temperature. As a further alternative, the temperature sensor may take the form of an integrated circuit mounted on a printed circuit board. The chip is in contact with a wing of the sleeve which extends into the void region between the wells and which acts as a thermal mass for dynamic similarity with the solution temperature.
These and other features and advantages of the present invention will become apparent from the following detailed description of preferred embodiments which, taken in conjunction with the accompanying drawings, illustrate by way of example the principles of the invention.